JASEM ISSN 1119 -8362 Full-text Available Online at J. Appl. Sci. Environ. Manage. https://www.ajol.info/index.php/jasem Vol. 22 (5) 681 – 687 May 2018 All rights reserved http://ww.bioline.org.br/ja Ameliorative Potential of Biocharcoal on Sodium Azide Toxicity in African Yam Bean ( Hochst. Ex. A. Rich) Harms.

*OSHOMOH, EO; ILODIGWE, CC

Department of Science Laboratory Technology, Faculty of Life Sciences, University of Benin, Benin, Nigeria *Corresponding Author Email [email protected] ; +2348055452141

ABSTRACTS: The study focused primarily on evaluating the ameliorative potential of biocharcoal on sodium azide toxicity (35 ppm) in African yam bean ( Sphenostylis stenocarpa Hochst. Ex. A. Rich) Harms. A field experiment was conducted using river sand and mixtures of biocharcoal from Pentaclethra macrophylla , represented by 0 (100 % river sand, control), 25, 50, 75 and 100 % biocharcoal added to ameliorate the toxic effects of sodium azide on African yam bean. Morphological parameters measured include germination percentage, number of leaves and secondary roots, percentage seedling emergence, shoot height, number of branches and number of leaves were recorded. The effect of biocharcoal on the toxicity of sodium azide to soil pH, conductivity, nitrogen, phosphorus, potassium contents and microbial community were also recorded. The growth of African yam bean was significantly improved by the application of the biocharcoal as higher height, number of leaves, percentage seedling emergence and number of branches were observed when compared with control. Soil conductivity, potassium and phosphorus were significantly higher in the treatments with biocharcoal. No positive effect on soil nitrogen content was observed. Biocharcoal addition adversely affected soil microbial community. Biocharcoal proved to have ameliorative potential however more work is needed to understand the mechanism by which it operates.

DOI: https://dx.doi.org/10.4314/jasem.v22i5.11

Copyright: Copyright © 2018 Oshomoh and Ilodigwe. This is an open access article distributed under the Creative Commons Attribution License (CCL), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Dates : Received: 11 March 2018; Revised: 09 April: 2018; Accepted: 25 April 2018

Keywords: Mutants, sodium azide, biochacoal, microbial community

With increase in world population of humans, food properties/quality and enhancing significant increase security and its sustainability become a global in crop yields thus promoting plant productivity concern. Chemical mutagens, one of the three types of (Mensah and Ekeke, 2016). The addition of mutagens, has been shown to create mutant of biocharcoal on the soil can improve soil properties and desirable quality for example sodium azide (NaN 3) on have other environmental benefit (Thi et al ., 2013). cowpea, NaN 3 on rice, NaN 3 on maize. Irrespective of Charcoal improves nutrient retention capacity and these positive results obtained from chemical increase soil fertility (Glaser et al ., 2002). The mutagenesis in plant and adverse effect of these improved nutrient retention may also lead to less mutagen from crop have also been reported. This nutrient leaching. Applying charcoal to the soil can therefore forms the basis of this research. Irrespective improve water holding capacity (Chan et al ., 2007). of the aforementioned advantages, mutation breeding Charcoal usually increases soil pH because it contains with the use of chemical mutagens could be some ash which can act as liming agent as well as detrimental to the health and yield of (Khan and phosphorus (P), potassium (K) fertilizer (Thi et al ., Al-Quarainy, 2009) and this should be taken seriously 2013). African yam bean ( Sphenostylis stenocarpa because mutagens can be found in the environment as Hochst. Ex. A. Rich) is an herbaceous leguminous pesticides, food additives, industrial chemicals and plant occurring throughout tropical Africa. African industrial effluents, hence this research to investigate yam bean is an underutilized species with great genetic the effectiveness of Bio-charcoal in ameliorating the and economic potential, African yam bean is one of possible negative effects of the mutagen, Sodium many indigenous food crops of Africa which have azide, on the test plant African yam bean ( Sphenostylis tendency to ameliorate nutritional food insecurities but stenocarpa (Hochst. Ex. A. Rich) Harms). it is neglected due to poor awareness and limited research about the , agronomy, genetics, Biocharcoal is a material produced by the medicinal value and productive potentials of the crops. thermochemical pyrolysis of biomass material. It can The subsistence production of the crop may have been be used in soil amendment for improving soil occasioned by the poor acceptability as a valuable crop

*Corresponding Author Email [email protected] ; +2348055452141

Ameliorative Potential of Biocharcoal on ….. 682 among middle aged farmers in Africa (Adewale et al ., to ANOVA using the SPSS version 16.0 software 2012) and its low consumption rate is mainly due to its (Ogbeibu, 2005). long cooking time. The generic name Sphenostylis RESULTS AND DISCUSSION arose from a greek word "Sphen" meaning wedge The result of the effect of charcoal on the percentage shape. Therefore, the genus Sphenostylis comprises a seedling emergence of African yam bean subjected to group of leguminous species with dorsiventrally sodium azide pollution is presented in table 1. The cuncate style with flattened stigmatic tip. Former addition of biocharcoal had no positive effect at the grouping within the two genera was because they were first two weeks but significantly higher percentage found to be closely related to them hence most species seedling emergence was recorded at the third week in the genus Sphenostylis initially bear Dolchius and after planting in the treatments with biocharcoal Vigna synonyms (Adewale et al ., 2012). African yam amendment (Table 1). The chemistry of azide's bean belongs to the order and the family reaction with biocharcoal or carbon compounds is still . a current interest of researchers (Halbig et al ., 2015) however there is no documentation to the best of my The objective of paper is to investigate the knowledge on the possible effect biocharcoal can have ameliorative potentials of biocharcoal on sodium azide on mutagenicity. So, the mechanism responsible for toxicity (35ppm) in African yam bean (Sphenostylis the result seen in table 1 of this study is farfetched. stenocarpa Hochst. Ex. A. Rich) Harms However, biocharcoal has been proven to have ameliorative potentials as it can adsorb toxic MATERIALS AND METHODS compounds (Brendova et al ., 2015; Cui et al ., 2011) Experimental Site : This experiment was carried out in and this supports the results of this study. Test subjects the Botanical Garden of the Department of Plant in the treatment with biocharcoal amendment recorded Biology and Biotechnology, Faculty of Life Sciences, higher plant height than those of the control treatment University of Benin. The seeds of African yam bean and this was observed from the first week to the sixth (Umuidike local) were purchased from a local market week after treatment. No growth was recorded in the in Umuidike, Abia State. The sodium azide chemical control treatment. The results seen in table 2 of this used for this experiment was purchased from Mosdelic study is in agreement with that of Pluchon et al . (2014) Chemical Store, University of Benin, Benin City. who reported that charcoal has ameliorative properties River sand utilized for this experiment was obtained and can act as an adsorbent for negative compounds. from a construction site within the University of The lack of growth observed in the control treatment Benin’s premises. The charcoal utilized was purchased is in agreement with Akhtar (2014) than mutagens can from a saw mill in Ovbiogie, Ovia North East Local have adverse effects on shoot height. The result in Government, Edo State. table 2 of this study is also in agreement with the report by Mensah and Obadoni (2007) who reported a The experiment consisted of five sets of treatments concentration dependent adverse effect of sodium each having six replicates. The first set contained 100 azide on the shoot height of Arachis hypogaea L. The % river sand to 0 % biocharcoal. The second set exact mechanism by which biocharcoal boosted the contained 75 % river sand to25 % biocharcoal. The shoot growth of African yam bean subjected to sodium third contained equal proportion of river sand and azide pollution is yet to be understood, however biocharcoal. The fourth had a combination of 25 % besides ameliorative abilities biocharcoal has also sand to 75 % biocharcoal while the last set was made been reported to be a stimulant to plant growth (Roy et up of 100 % biocharcoal. Total weight of soil and al ., 2012). The result of the effect of biocharcoal on biocharcoal mixture contained 1500 g in each nursery the number of leaves of Sphenostylis stenocarpa bag. Each bag was irrigated with 35 mg/l NaN 3 subjected to sodium azide pollution is presented in this regularly.The charcoal used was made from the wood table 3. Test plants in the treatment with the of Pentaclethra macrophylla commonly called biocharcoal amendment had more number of leaves Okpagha in Edo state and ûkpag ā in the Eastern part than the control treatment, in which no leaf growth was of Nigeria. Physiological and morphological recorded. In table 3, it is seen that no leaf was parameters like the seedling emergence percentage, produced in the control treatment as sodium azide at number of leaves and secondary roots were recorded 30 mg/l concentration significantly inhibited the in the laboratory and plant height, number of leaves growth of African yam bean. This is supported by the and number of branches were recorded in the field result of Gnanamurthy et al . (2012) who reported the experiment. Soil used for the experiment was adverse effect of sodium azide on the leaf growth of subjected to chemical analysis and microbial analysis Zea mays . The boost in leaf production by biocharcoal at the tail end of the experiment. The result obtained is supported also by Herath et al . (2015); Diaz- from the experiment was arranged and then subjected Muegue et al . (2016) and Mensah and Ekeke (2016)

OSHOMOH, EO; ILODIGWE, CC Ameliorative Potential of Biocharcoal on ….. 683 who all stated that biocharcoal can sustain the growth presented in table 4. No branches were observed in the of plants under duress. The result of the effect of control treatment as no growth was observed there but biocharcoal on the number of branches of Sphenostylis treatments with the biocharcoal amendments recorded stenocarpa subjected to sodium azide pollution is significantly higher number of branches.

Table 1 : Effects of biocharcoal on the percentage seedling emergence of Sphenostylis stenocarpa grown under sodium azide pollution. TREATMENTS TIMES (WEEK) 1 2 3 100 % soil (control) 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 25 % charcoal + 75 % soil 15.00±2.89 a 16.67±6.67 a 23.33±1.67 b 50 % charcoal + 50 % soil 11.67±10.41 a 20.00±11.55 a 20.00±5.00 b 75 % charcoal + 25 % soil 16.67±8.82 a 16.67± 8.82 a 25.83±8.70 b 100 % charcoal 3.33±3.33 a 11.67±1.67 a 15.00±8.70 b P-value 0.169 0.402 0.022 Significance (at P ≤0.05) NS NS S S= significant; NS= not significant; Level of significance = 0.05; Duncan multiple range test (mean comparison) = mean ± standard error a Table 2: Effects of biocharcoal on the shoot height of Sphenostylis stenocarpa grown on sodium azide polluted soil. TREATMENTS TIMES (WEEK) (cm) 3 4 5 6 100 % soil (control) 0.00 ± 00 a 0.00± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 25 % charcoal + 75 % soil 9.75 ± 0.90 b 10.22± 1.48 b 9.75 ± 4.88 b 5.67 ± 5.67 a, b 50 % charcoal + 50 % soil 10.96 ± 3.71 b 9.31 ± 0.78 b 12.26± 0.75 b 13.50 ± 0.58 b, c 75 % charcoal + 25 % soil 10.34 ± 0.77 b 11.72± 1.64 b 15.27 ± 1.67 b 15.04 ± 0.81 c 100 % charcoal 9.24 ± 1.05 b 11.64 ± 0.95 b 13.13 ±0.91 b 16.02 ± 1.12 c P-value 0.000 0.000 0.008 0.006 Significance (at P ≤0.05) S S S S S= significant; NS= not significant; Level of significance = 0.05; Duncan multiple range test (mean comparison) = mean ± standard error

Table 3 : Effects of biocharcoal on number of leaves of Sphenostylis stenocarpa grown on sodium azide polluted soil. TREATMENTS TIMES (WEEK) 3 4 5 6 100 % soil (control) 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 25 % charcoal + 75 % soil 6.00±0.50 b 2.67±2.67 a 4.00±2.00 b 3.00±3.00 ab 50 % charcoal + 50 % soil 3.50±0.87 b 8.78±1.77 b 11.67±1.20 cd 8.83±0.17 bc 75 % charcoal + 25 % soil 4.92±0.65 b 8.36±1.87 b 7.81±0.81 bc 10.12±1.77 c 100 % charcoal 5.50±1.32 b 11.08±0.87 b 12.94±1.38 d 13.50±3.28 c P-value 0.002 0.012 0.015 0.002 Significance (at P ≤0.05) S S S S S= Significant; NS= not significant; Level of significance = 0.05; Duncan multiple range test (mean comparison) = mean ± standard error a Table 4: Effects of biocharcoal on number of branches of Sphenostylis stenocarpa grown on sodium azide polluted soil. TREATMENTS TIMES (WEEK) 3 4 5 6 100 % soil (control) 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a 25 % charcoal + 75 % soil 2.33±0.33 b 4.00±00 c 1.33±0.67 a,b 1.00±1.00 a,b 50 % charcoal + 50 % soil 1.33±0.33 b 0.00±0.00 a 4.00±0.58 b 3.00±0.00 b,c 75 % charcoal + 25 % soil 1.67±0.33 b 0.75±0.48 a 3.00±0.00 b,c 3.67±0.67 c 100 % charcoal 2.00±0.58 b 2.50±0.29 b 4.33±0.88 c 4.67±1.20 c P-value 0.000 0.012 0.000 0.003 Significance (at P ≤0.05) S S S S S= Significant; NS= not significant; Level of significance = 0.05; Duncan multiple range test (mean comparison) = mean ± standard error a From table 4 it shows that growth didn't occur in the The result of the effect of charcoal on the pH of the control treatment and this is supported by the result soil polluted with sodium azide is presented in figure obtained by Nakweti et al . (2015) who reported that 1. The soil pH did not follow any particular trend sodium azide can have adverse effect on branch however the highest pH was obtained in the treatment formation in plants. The boost in branch production by with 50 % biocharcoal while the lowest pH value was biocharcoal on African yam bean subjected to sodium recorded in the treatment with 25 % biocharcoal. azide toxicity as shown by the result on table 4 in this Gruszka et al . (2012) reported that sodium azide study is supported by Herath et al . (2015), Diaz- toxicity is affected by soil pH. This makes it important Muegue et al . (2016) and Mensah and Ekeke (2016) to know the effect of biocharcoal on soil pH. who all confirmed that biocharcoal can soak up toxic substances and may possess ameliorative properties.

OSHOMOH, EO; ILODIGWE, CC Ameliorative Potential of Biocharcoal on ….. 684

Fig 1: Effect of biocharcoal on pH of soil polluted with sodium Fig 3: Effect of biocharcoal on Nitrogen content of soil polluted azide. with sodium azide.

The result of this study as seen in figure 1 agrees with Thi et al . (2013) says biocharcoal can't replace the report by Zhang et al . (2016) who reported that fertilizer as nitrogen is highly needed for plant growth. biocharcoal addition increased soil pH. However, the Mitchual et al . (2014) also reported that biocharcoal’s increase reported in this study was only observed at 25 effect on nitrogen varies with the source of the wood % Biocharcoal treatment. No peculiar trend was used to prepare it. observed when taking all treatments into perspective. It is important to note that the high pH in the control treatment is in agreement with the report by Chefetz et al . (2006) that sodium azide can elevate soil pH. The effect of biocharcoal amendment on soil conductivity is presented in figure 2. The result of the conductivity followed an interesting trend. The results show that as the proportion of charcoal used increases so did the conductivity of the soil.

Fig 4: Effect of biocharcoal on phosphorus content of soil polluted with sodium azide.

The effect of biocharcoal on the soil phosphorus content of soil polluted with sodium azide is presented in the folowing figure 4. The soil phosphorus content was shown to reduce with increase in the proportion of biocharcoal utilized.

Fig 2: Effect of biocharcoal on conductivity of soil polluted with The result gotten from figure 4 of this study is not in sodium azide. agreement with the reports of Lehmann et al . (2011); Nabavinia et al. (2015) and Thi et al . (2013), who The result in figure 2 of this study is in agreement with reported that biocharcoal addition can increase the the report by Nabavinia et al . (2015) and Zhang et al . phosphorus content of the soil as the phosphorus (2016) who reported that biocharcoal addition led to content didn't increase when soil was amended with elevated soil conductivity. Hence, in this study better biocharcoal. Biocharcoal has been referred to as a growth performance was recorded in soil with elevated potassium fertilizer by Thi et al . (2013). Result of this conductivity. The effect of biocharcoal amendment on effect of biocharcoal amendment on potassium content the nitrogen content of soil polluted with sodium azide of soil polluted with sodium azide is presented in table is shown in figure 3. Treatments with 25, 50, 75 and 5. The soil potassium content was shown to increase 100 % had lower soil nitrogen content compared to the in direct proportion to the percentage biocharcoal control treatment which had the highest nitrogen applied. The control had the least potassium soil content. In figure 3 has shown the effect of biocharcoal content while the treatment with 100 % biocharcoal on soil nitrogen content of a soil polluted with sodium had the highest soil potassium content. This agrees azide however it is relevant to note that biocharcoal with the report from figure 5 of this study as potassium additions has been reported to increase soil nitrogen was observed to increase with increase in biocharcoal content (Nigussie and Kissi, 2011). proportion used. Chefetz et al . (2006) reported sodium azide to be biocidal on bacterial and fungal population.

OSHOMOH, EO; ILODIGWE, CC Ameliorative Potential of Biocharcoal on ….. 685

treatments with biocharcoal can't be fully elucidated as there is no visible literature on the effect of the interaction of biocharcoal and sodium azide on bacteria colony count.

Fig 5: Effect of biocharcoal on potassium content of soil polluted with sodium azide.

Sreeju et al . (2011) reported the contrary. More work is needed to understand the reason for the differences in reports. Adeniyi (2010) supported that biocharcoal could have an effect on microbial community. Bacteria count of sodium azide polluted soil amended with biocharcoal is presented in figure 6. Treatment Fig 6: Effect of biocharcoal on bacteria colony count of soil polluted with 25 % and 50 % Biocharcoal significantly boosted with sodium azide. bacteria colony count but the value was shown to reduce as the proportion of biocharcoal used reduced. Results of the effect of biocharcoal amendment on The control treatment had the second lowest bacteria specific bacteria of sodium azide polluted soil is colony count. Lehmann et al . (2011) reported that presented in table 5. Biocharcoal amendment affected biocharcoal can increase microbial biomass and the soil bacteria composition as the control treatment had result from figure 6 agree with this report. The boost more of the specific isolates screened than the in bacteria colony count as shown by the result from treatments with the biocharcoal amendment. The figure 6 of this study is also in agreement with the result also showed that 25 % biocharcoal amendment report by Meynet et al . (2002). The decline in bacteria had equal effect as the control treatment making it less colony count recorded in the ultimate and penultimate of a biocide.

Table 5: Effect of biocharcoal on the specific bacteria presence in the screening of soil treated with sodium azide

Table 6: Effect of biocharcoal on the specific fungi presence screening of soil treated with sodium azide Samples (30 Ppm) Isolates 0 % Aspergillus flavus, Trichoderma harzianum, Mucor mucedo, Fusarium solani, Aspergillus niger 25 % Trichoderma harzianum, Aspergillus flavus, Mucor mucedo, Aspergillus niger 50 % Aspergillus flavus, Aspergillus niger 75 % Aspergillus niger, Mucor mucedo 100 % Mucor mucedo, Aspergillus niger

The result of the effect of charcoal application on the control treatment, the treatment with 30 ppm NaN 3 had microbial fungi colony count of soil treated with the highest fungal colony count. The result from figure sodium azide is presented in figure 7. The control 7 is also in agreement with the report of Gao et al . treatment had the highest fungi colony count while the (2016) who states that biocharcoal can have varying treatment with 100 % biocharcoal amendment had the effect on fungal population and can also tilt the odds least fungi colony count. in favor of the bacteria population. The result of the effect of charcoal on specific fungi presence in the Sodium azide was reported to negatively affect fungal screening of soil polluted with sodium azide is population by Kumi et al . (2013) and the result from presented in table 6. figure 7 of this study disagrees with this report as the

OSHOMOH, EO; ILODIGWE, CC Ameliorative Potential of Biocharcoal on ….. 686

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Akhtar, N (2014). Effect of physical and chemical behavior of tomato ( Lycopersicon esculentum L.) cv. “Rio grande” under heat stress conditions. Schol. J. Agr. Sci. 4: 350-355.

Brendova, K; Thistos, P; Szakova, J (2015). Biochar immobilizes cadmium and zinc and improves phyto extraction potential of Willow plants on extremely contaminated soil. Pl. S. Environ. 61: 303-308. Fig 7: Effect of biocharcoal on fungi colony count of soil polluted with sodium azide. Chan, KY; Zwieten, LV; Meszaros, I; Downie, A; The presence of the specific fungi isolates dwindled as Joseph, S (2007). Agronomic values of the proportion of biocharcoal used increased however greenwaste biochar as a soil amendment. Austr. J. there was no difference in the presence of the specific S. Res. 45: 629-634 fungi isolates in 25 % biocharcoal treatment and the control treatment. From table 5 and 6, it can be seen Chefetz, B; Stinler, K; Scechter, M; Drori, Y (2006). that Biocharcoal at proportions above 25 % adversely Interactions of sodium azide with triazine affected the presence of the bacteria and fungi herbicides: effect of sorption to soils. screened. This change, the alteration as it is in Chemosphere 65: 352-357. agreement with the report by Adeniyi (2010); Lehmann et al . (2011) and Gao et al . (2016) who Cui, L; Li, L; Zhang, A; Pan, G; Bao, D; Chang, A reported that biocharcoal can alter microbial (2011). Biochar amendment greatly reduces rice community structure. The result from table 5 and 6 Cd uptake in a contaminated paddy soil: a two- differs from report by Sreeju et al . (2011) and Meynet year field experiment. BioRes. 6: 2605-2618. et al . (2002) who stated that biocharcoal had no effect on soil fungi as the result showed a decline in fungi Diaz-Muegue, L; Pino, N; Penuela, G (2016). Biochar species presence with elevation in proportion of from oil palm waste as an amendment for the charcoal used. remediation of soil disturbed by open-cast coal mining. Glob. Res. J. Eng. Techno. Innov. 5: 017- Conclusion : Sodium azide may be beneficial for the 022. production of mutant plants with desirable traits but it is not without adverse effects and not until this Gao, X; Cheng, HY; Valle, ID; Liu, S; Masiello, C; negative effect is controlled, the advantages of Silberg, JJ (2016). Charcoal disrupts soil chemical mutagenesis by sodium azide will be microbial communication through a combination dampened by its adverse effect. Biocharcoal proved to of signal sorption and hydrolysis. Amer. Chem. be a biomaterial with ameliorative potential, able to Soc. Ome. 1: 226-233. mitigate the adverse effects of sodium azide however this can’t be conclusive until its mechanism of action Glaser, B; Lehmann, J; Zech, W (2002). Ameliorating is understood. physical and chemical properties of highly weathered soils in the tropics with charcoal. A REFERENCES Rev. Biol. Fert. Soi. 35: 219-230.

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OSHOMOH, EO; ILODIGWE, CC